JP2008002948A - Detection method of target nucleic acid, and container used for detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of detecting target nucleic acids simply and with high accuracy, without having to utilize hybridization method, and to provide a container used for the detection method. <P>SOLUTION: This detection method of the target nucleic acid 30, formed by bonding a first ligand 31 and a second ligand 32 of each different kind to a nucleic acid of a detection object, has a process for preparing a first particle 33 to which a plurality of first receptors 331, to be bonded specifically to the first ligand 31, are bonded, and a second particle 34 to which a plurality of second receptors 341 to be bonded specifically to the second ligand 32 are bonded; a process for mixing a sample, having a possibility of containing the target nucleic acid 30, the first particle 33 and the second particle 34 in the container, having a sediment capturing part on the bottom surface; and a process for observing the captured distribution on the container bottom surface, of an aggregate comprising the target nucleic acid 30, the first particle 33 and the second particle 34 which are connected together and/or the sediment of the first particle 33 and the second particle 34, after the mixing process. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting the presence or absence or concentration of a nucleic acid to be detected and a container used for the detection method.

  As a typical technique for detecting a specific target nucleic acid present in a sample, a technique using a hybridization method is known (see Patent Document 1). This method is a technique for detecting a very small amount of DNA or RNA target nucleic acid with a probe from a sample containing many kinds of nucleic acids.

On the other hand, a method of detecting a specific target molecule present in a sample by agglutinating is also known. Various containers have been proposed so far that perform aggregation reaction by this method and detect the resulting aggregate. For example, a container that can visually determine the aggregate pattern of the aggregate is proposed. (See Patent Document 2).
Japanese Unexamined Patent Publication No. 7-51099 Japanese Patent Publication No. 63-60854

However, nucleic acids may hybridize non-specifically. For example, when the target nucleic acid has a similar base sequence to the non-target nucleic acid, it is difficult to selectively detect only the target nucleic acid in the sample. Therefore, the method described in Patent Document 1 has a problem that the target nucleic acid cannot always be detected with high accuracy.
In addition, the container described in Patent Document 2 is used for detection of red blood cells, and since nucleic acids and red blood cells have a large difference in structure including molecular size, they cannot be directly used for detection of target nucleic acids. there were.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for detecting a target nucleic acid simply and with high accuracy without using a hybridization method, and a container used for the detection method. .

To solve the above problem,
The invention according to claim 1 is a method for detecting a target nucleic acid, in which a first ligand and a second ligand of different types are bound to a nucleic acid to be detected, which specifically binds to the first ligand. Preparing a first microparticle having a plurality of first receptors bound thereto and a second microparticle having a plurality of second receptors bound specifically to the second ligand; and In a container having a sediment trap, a sample that may contain the target nucleic acid, a step of mixing the first microparticles and the second microparticles, and a first ligand after the mixing step An aggregate comprising the target nucleic acid and the first microparticles and the second microparticles linked via the binding of the first receptor and the binding of the second ligand and the second receptor, respectively, and / or Precipitation of the first and second particulates Of things, a method for detecting a target nucleic acid comprising a step of observing a capturing distribution in the bottom of the container.
The invention according to claim 2 is a method for detecting a target nucleic acid, in which a first ligand and a second ligand of different types are bound to a nucleic acid to be detected, which specifically binds to the first ligand. Preparing a container in which a plurality of first receptors are bound to the bottom surface, preparing magnetic fine particles in which a plurality of second receptors that specifically bind to the second ligand are bound, and A step of adding the sample possibly containing the target nucleic acid and the magnetic fine particles in the container, and after the adding step, applying a magnetic force to the magnetic fine particles from the outside of the container A method for detecting a target nucleic acid, comprising the step of observing the distribution of magnetic fine particles in
The invention according to claim 3 is a method for detecting a target nucleic acid, characterized in that both the detection method according to claim 1 and the detection method according to claim 2 are performed.
Invention of Claim 4 is the observation result when using the sample (1) containing the target nucleic acid of known concentration in the method for detecting a target nucleic acid according to any one of Claims 1 to 3 And the observation result when using the sample (2) that does not contain the target nucleic acid and the observation result when using the sample (3) that may contain the target nucleic acid And detecting the presence or concentration of the target nucleic acid in the sample (3).
The invention according to claim 5 is the method for detecting a target nucleic acid according to any one of claims 1 to 3, and an observation result when using a sample containing a known different concentration of the target nucleic acid. Comparing the observation result when using the sample (4) that may contain the target nucleic acid, and confirming the presence or concentration of the target nucleic acid in the sample (4), This is a method for detecting a target nucleic acid.
The invention according to claim 6 is characterized in that the ligand is a hydrophilic organic compound, digoxigen, fluorescein, alexa, fluorescein isothiocyanate (FITC), 2,4-dinitrophenol (DNP), tetramethylrhodamine (TAMRA), A polypeptide comprising a polypeptide having 6 or more amino acid residues, a sugar chain in which two or more monosaccharides are bound, biotin, protein, polyhistidine, HA, glutathione S-transferase (GST), and an amino acid sequence represented by SEQ ID NO: 1. The method for detecting a target nucleic acid according to any one of claims 1 to 5, wherein the method is selected from the group consisting of (Flag) and derivatives thereof.
In the invention according to claim 7, the target nucleic acid is prepared by a polymerase chain reaction method using a first primer to which a first ligand is bound and a second primer to which a second ligand is bound. The method for detecting a target nucleic acid according to any one of claims 1 to 6, wherein the target nucleic acid is detected.
The invention according to claim 8 is characterized in that the target nucleic acid incorporates at least one nucleotide bound to the first ligand using the first primer, and further uses the second primer to The method for detecting a target nucleic acid according to any one of claims 1 to 6, wherein the target nucleic acid is prepared by a polymerase chain reaction method while incorporating at least one nucleotide having a ligand bound thereto. is there.
The invention according to claim 9 is characterized in that the target nucleic acid is of a plurality of different types, and at least one of the first and second ligands in the target nucleic acid is different for each type of target nucleic acid. Item 9. The method for detecting a target nucleic acid according to any one of Items 1 to 8.
The invention according to claim 10 is the method for detecting a target nucleic acid according to any one of claims 1 to 9, wherein the target nucleic acid is derived from a living body.
The invention according to claim 11 is characterized in that the first and second fine particles have a particle size of 0.1 to 10 μm and are optically distinguishable from each other. It is a detection method of the target nucleic acid as described in any one.
The invention according to claim 12 is characterized in that the plurality of containers are provided on the same plate, and the capacity of each container is 0.1 to 0.5 ml. The method for detecting a target nucleic acid according to one item.
In the invention according to claim 13, the sediment trapping part of the container is formed by concentrically arranging a plurality of concavities and convexities having a sawtooth shape in a radial cross section on a bottom surface having a mortar shape. The length (h) of the first inclined surface that connects the valley from the mountain to the outside is 0.2 to 20 μm, and the length (l) of the second inclined surface that connects the mountain to the mountain from the center to the outside is (l). It is 0.5-50 micrometers, and the angle ((theta) ₁ ) which the straight line which connects a mountain and a center makes with a horizontal surface is 15-45 degrees, It is any one of Claim 1 and 3-12 characterized by the above-mentioned. It is the detection method of the target nucleic acid of description.
In the invention according to claim 14, the sediment trapping part of the container is provided with a plurality of recesses independently provided on a bottom surface having a mortar shape, and an angle (θ ₂₎ between the bottom surface and a horizontal plane. ) Is 15 to 45 °, the method for detecting a target nucleic acid according to any one of claims 1 and 3-12.
The invention described in claim 15 is characterized in that the recess has a diameter (L) of an opening of 0.05 to 100 μm and a depth (d) of 0.03 to 60 μm. This is a method for detecting a target nucleic acid.
The invention according to claim 16 is a method for detecting a target nucleic acid, characterized in that both the detection method according to claim 13 and the detection method according to claim 14 or 15 are performed.
The invention according to claim 17 is the method for detecting a target nucleic acid according to any one of claims 2 to 10 and 12, wherein the magnetic fine particles have a particle size of 0.1 to 20 μm. .
The invention described in claim 18 is characterized in that the container has a substantially hemispherical surface or a substantially conical surface at the bottom, and an angle (θ ₃ ) between the bottom and the horizontal surface is 15 to 45 °. A method for detecting a target nucleic acid according to any one of claims 2 to 10, 12 and 17.

The invention according to claim 19 is a container used in the method for detecting a target nucleic acid according to any one of claims 1 and 3 to 12, wherein the radial cross section is serrated in a mortar-shaped bottom surface. Are arranged in a concentric manner, the length (h) of the first inclined surface connecting the mountain to the valley from the center to the outside is 0.2 to 20 μm, the valley to the mountain from the center to the outside The length (l) of the second inclined surface connecting the two is 0.5 to 50 μm, and the angle (θ ₁ ) between the straight line connecting the mountain and the center and the horizontal plane is 15 to 45 °. A target nucleic acid detection container.
The invention according to claim 20 is a container used in the method for detecting a target nucleic acid according to any one of claims 1 and 3 to 12, wherein a plurality of recesses are independently formed on a bottom surface having a mortar shape. The target nucleic acid detection container is characterized in that the angle (θ ₂ ) between the bottom surface and the horizontal plane is 15 to 45 °.
The invention according to claim 21 is characterized in that the opening has a diameter (L) of 0.05 to 100 μm and a depth (d) of 0.03 to 60 μm. This is a container for detecting a target nucleic acid.
The invention according to claim 22 is a container used in the method for detecting a target nucleic acid according to any one of claims 2 to 10, 12 and 17, wherein the bottom surface is substantially hemispherical or substantially conical. The target nucleic acid detection container is characterized in that the angle (θ ₃ ) between the bottom surface and the horizontal plane is 15 to 45 °.
The invention described in claim 23 is a target nucleic acid detection container, wherein a plurality of containers according to any one of claims 19 to 22 are provided on the same plate.

  According to the present invention, the presence or concentration or concentration of the target nucleic acid can be detected easily and with high accuracy by visually observing the sediment capture distribution on the bottom surface of the container.

Hereinafter, the present invention will be described in detail.
The target nucleic acid detection method of the present invention is roughly divided into a step of obtaining a target nucleic acid having a structure in which a ligand is bound to a nucleic acid to be detected, and a step of detecting the target nucleic acid. Hereinafter, each step will be described in detail.

<Step of obtaining target nucleic acid>
First, the process for obtaining the target nucleic acid will be described.
In the present invention, the nucleic acid to be detected is not particularly limited, and is any nucleic acid or nucleic acid analog including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA, synthetic RNA, etc., and is derived from a living body. Or it may be artificially synthesized.

  A conventionally known ligand can be used as the ligand to be bound to the nucleic acid to be detected. Preferred examples include hydrophilic organic compounds, haptens, digoxigen, fluorescein, alexa, fluorescein isothiocyanate (FITC), 2 , 4-dinitrophenol (DNP), tetramethylrhodamine (TAMRA), polypeptide having 6 or more amino acid residues, sugar chain to which 2 or more monosaccharides are bonded, biotin, protein, polyhistidine, HA, glutathione S-transferase (GST), a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 (Flag), and derivatives thereof can be used, but are not limited thereto. Biotin, digoxigen, fluorescein, Alexa, etc. have the advantage of being easily available from the market and low in price. In addition, sugar chains and peptides are advantageous in that they have a high degree of freedom in the design of chemical structures and can be easily arranged in multiple types. What is necessary is just to select the ligand to be used suitably by the nucleic acid, sample, etc. of detection object.

  One or more different types of first and second ligands are bound to the target nucleic acid. As shown in FIG. 1, for example, the method for obtaining a target nucleic acid having a structure in which the first and second ligands are bound to the nucleic acid to be detected can be obtained by using (i) light or platinum for the nucleic acid 10 to be detected. Alternatively, a method of covalently binding the first ligand 31 and the second ligand 32 to the base and / or the 5 ′ end of the nucleic acid via a linker (FIG. 1A), for example, the 5 ′ end of the nucleic acid A method of covalently bonding the amino group of the base and the carboxyl group of the ligand, (ii) a first primer 11 to which the first ligand 31 is bound during nucleic acid synthesis, and a second ligand 32 to which the second ligand 32 is bound. A method of extending a base sequence by polymerase chain reaction (PCR) method using the second primer 12 (FIG. 1 (b)), (iii) nucleotide to which the first ligand 31 is bound in the PCR method A method of extending the base sequence while incorporating at least one of 3 and at least one nucleotide 14 to which the second ligand 32 is bound (FIG. 1 (c)), (iv) using terminal deoxyzyltransferase Various methods such as a method of tailing the nucleotide to which the first ligand 31 or the second ligand 32 is bound to the 3 ′ end of the nucleic acid (FIG. 1D) can be used.

  For samples in which the target nucleic acid is present together with other nucleic acids, such as biological samples or arbitrary analytes, the methods (ii) and (iii) are preferred. Any method may be used when the target nucleic acid is present alone. For example, a nucleic acid to be detected contained in a biological sample or the like may be detected after being amplified by a PCR method, but may be used as it is without being amplified.

  As shown in FIG. 1, there are two types of ligands in the target nucleic acid 30, a first ligand 31 and a second ligand 32. The structure of the nucleic acid is flexible, and in the present invention, as described later, it is essential that the target nucleic acid binds to different types of first and second microparticles.

  Further, the number of the first ligand and the second ligand in the target nucleic acid may be one each, but may be two or more. Since the volume ratio of nucleic acid to microparticle is extremely large, the possibility that three or more microparticles bind to one nucleic acid is low. Therefore, even if there are a plurality of ligands in one target nucleic acid, it is considered that the aggregation degree of the fine particles to which the target nucleic acid is bound is not affected.

  Moreover, when there are a plurality of types of target nucleic acids, for example, the method (ii) or (iii) may be performed simultaneously to prepare a plurality of types of target nucleic acids. In this case, at least one of the first ligand and the second ligand in each target nucleic acid is preferably different for each type of target nucleic acid.

<Step of detecting target nucleic acid>
Next, the process for detecting the target nucleic acid will be described.
The detection process is roughly divided into two methods, and it is possible to perform detection alone, but it is possible to obtain a detection result with higher accuracy by combining the two methods. The two methods will be described below in order.

[First method]
(Step of preparing first fine particles and second fine particles)
A first microparticle in which a plurality of first receptors that specifically bind to the first ligand are bound, and a second microparticle in which a plurality of second receptors that specifically bind to the second ligand are bound To prepare.
The fine particles used in the present invention are dispersible fine particles. For example, fine particles that are dispersed in a solution such as colloid particles, and latex particles are preferably used, but are not limited thereto. The particle diameters of the first fine particles and the second fine particles are preferably 0.1 to 10.0 nm. If fine particles having such a particle size are used, a stable aggregate can be easily obtained.

  In the present invention, specific binding means binding based on intermolecular force with high specificity that is selectively formed between specific substances such as antigen and antibody.

  Latex particles are mainly composed of polystyrene, and methacrylic acid is copolymerized to enhance hydrophilicity and dispersibility. Latex particles include a plain type having no functional group on the surface and a functional group type having a functional group. The charge on the plain type surface has a negative charge due to the presence of methacrylic acid, and can ionically bond with the positively charged region in the protein molecule. It can also be hydrophobically bound to protein molecules. Since plain type latex particles adsorb proteins simply by mixing with proteins, protein immobilization is easy. Various types of receptors can be used as will be described later. When various proteins are used, such plain type latex particles are preferably used as fine particles.

Latex particles having a functional group are designed such that a carboxy group or an amino group is exposed on the surface, and various types of functional group-type latex particles can be used. When the receptor is bound to the functional group type latex particles, the functional group in the functional group type latex particles may be bound to the functional group of the receptor, and conventionally known methods can be applied, for example, EDAC method of combining carboxylic acid and amino group with water-soluble carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are mixed in advance and carboxylic acid And a method of bonding amino groups to each other using a bipolar linker, a method of bonding activated aldehyde groups or tosyl groups to functional groups in the receptor, and the like. In the present invention, any conventionally known method may be used as long as the effects of the present invention are not impaired, but the EDAC method is particularly preferable. And when using various proteins as a receptor like a plain type latex particle, a functional group type latex particle is used suitably.
Note that, even with fine particles other than latex particles, the receptor can be bound by a conventionally known method according to the type of fine particles.

  For the first receptor bound to the first microparticle, a receptor that specifically binds to the first ligand in the target nucleic acid is used, and for the second receptor bound to the second microparticle, A receptor that specifically binds to the second ligand is used. Examples of the receptor include, but are not limited to, antibodies, lectins, streptavidin and the like. Of these, proteins, particularly antibodies, are preferably used.

  In the present invention, as one embodiment, a single kind of receptor is bound to one fine particle. In this embodiment, since at least two types of ligands are bound to the target nucleic acid, at least two types of microparticles are prepared for one type of target nucleic acid. That is, as shown in FIG. 2, the first fine particles 33 (FIG. 2A) to which the first receptor 331 that specifically binds to the first ligand 31 is bound, and the second ligand 32, A second microparticle 34 (FIG. 2B) to which a second receptor 341 that specifically binds is bound is prepared.

  As another embodiment of the present invention, two or more kinds of receptors may be bound to one fine particle. In this embodiment, among two or more types of ligands in one target nucleic acid, a receptor that specifically binds to one type of ligand and a receptor other than a receptor that specifically binds to the remaining types of ligands Are combined into one fine particle. That is, it is designed so that only one kind of two or more kinds of ligands in the target nucleic acid binds to one fine particle.

  The first fine particles and the second fine particles are preferably optically distinguishable from each other. Specifically, for example, it is preferable that the first fine particles and the second fine particles have different colors instead of the same color. In this way, it is easy to identify the fine particles at the operation stage, and even when self-aggregation occurs with one kind of fine particles when observing the aggregated image, it can be easily distinguished by color, and self-aggregation Is less likely to be misjudged as agglomeration by the first fine particles and the second fine particles. That is, the target nucleic acid can be detected with higher accuracy.

  The receptor binding to the microparticles in this step may be performed when detecting the target nucleic acid, but microparticles to which the receptor is bound in advance may be used for detection. Further, this step may be performed before or after the step of preparing the target nucleic acid.

(The step of mixing the sample that may contain the target nucleic acid, the first fine particles, and the second fine particles)
Next, a process of mixing a sample that may contain a target nucleic acid, the first fine particles, and the second fine particles will be described.
This step is performed in a container having a sediment capturing part on the bottom surface, which will be described later. If the sample that may contain the target nucleic acid to be used for detection actually contains the target nucleic acid, the binding between the first ligand and the first receptor as described above, and the second The target nucleic acid and the microparticle are linked through the binding between the ligand and the second receptor, and an aggregate is generated as described later. If the sample to be detected does not contain the target nucleic acid, such binding does not occur and aggregates do not occur.
This step may be performed by mixing the sample that may contain the target nucleic acid, the first microparticle, and the second microparticle at the same time, or the sample that may contain the target nucleic acid. On the other hand, the first fine particles and the second fine particles may be mixed in order and sequentially performed. The sample that may contain the target nucleic acid, the first microparticle, and the second microparticle are each preferably used as a solution.

  The solution used in this step is preferably a buffer solution, and more preferably a buffer solution that promotes aggregation. Examples of such a preferable buffer include a buffer having the following composition; 200 mM MOPSO (pH 7.4), 4% dextran, 100 mM NaCl, and 0.1% sodium azide. What is necessary is just to select suitably according to a kind. When different types of buffer solutions are mixed in this step, it is preferable to use the microparticles after washing with the different buffer solutions two to three times.

  When the amount of the first and second microparticles used for mixing is too large, the detection accuracy decreases. Therefore, it is preferable that the amount of the first and second microparticles is smaller than that of the target nucleic acid. On the other hand, the amount of fine particles is more preferably 0.01 to 0.5 w / v%.

  For example, when an antigen and an antibody are used as the ligand-receptor combination, this step is preferably performed at 0 to 37 ° C. for 5 to 30 minutes. In the case of other ligand-receptor combinations, a suitable temperature, time, and buffer may be appropriately selected according to the type of the combination.

FIG. 3 shows a state in which the target nucleic acid is linked to the first and second microparticles. In FIG. 3, the first ligand 31 in the target nucleic acid 30 is linked to the first microparticle 33 via the first receptor 331. Further, the second ligand 32 in the target nucleic acid 30 is linked to the second microparticle 34 via the second receptor 341. That is, one target nucleic acid 30 is connected to the first microparticle 33 and the second microparticle 34, whereby the first microparticle 33 and the second microparticle 34 are connected to each other. Furthermore, a plurality of target nucleic acids 30 are linked to one fine particle, and each of the target nucleic acids 30 is linked to other fine particles. As a result, a large number of fine particles are linked together, resulting in aggregation of the fine particles. In addition, the dotted line extended from the receptor in FIG. 3 represents the bound nucleic acid simply.
Thereby, if the target nucleic acid is contained in the sample, aggregation of the fine particles occurs.

(Process for observing the sediment distribution on the bottom of the container)
Subsequently, the sediment capture distribution on the bottom surface of the container is observed.
Here, the sediment refers to the aggregate and the first and second microparticles that have settled without being used for linking with the target nucleic acid. The target nucleic acid can be detected by this step. Here, the presence or absence of the aggregate is determined by the naked eye, image processing, or the like. For image processing, for example, a technique of analyzing image data captured by a CCD camera can be used.

Next, a specific method for detecting a target nucleic acid will be described in detail.
Using a sample (1) containing a target nucleic acid of known concentration to which a ligand is bound as described above, the target nucleic acid is mixed with fine particles according to the method described above, and the capture distribution of aggregates on the bottom of the container is determined. Observe and serve as a positive control. Further, the sample (2) not containing the target nucleic acid is mixed with the fine particles according to the above-described method, and the sediment capture distribution on the bottom surface of the container is observed to serve as a negative control. Then, the sample (3) that may contain the target nucleic acid is similarly mixed with the fine particles according to the method described above, and the sediment distribution on the bottom of the container is observed and compared with the positive control and negative control. Thus, the presence or concentration of the target nucleic acid in the sample (3) is confirmed. Specifically, if the capture distribution pattern when using the sample (3) matches or is similar to the positive control, it is judged as positive and the target nucleic acid is contained in the sample (3). It is confirmed that On the other hand, if the pattern of the capture distribution when using the sample (3) matches or is similar to the negative control, it is determined to be negative, and the target nucleic acid is contained in the sample (3). It is confirmed that there is not. On the other hand, when the pattern of the capture distribution when the sample (3) is used is intermediate between the positive control and the negative control, it is determined as a false positive. In this case, there is a possibility that a small amount of the target nucleic acid is contained in the sample (3). Thus, the presence or absence of the target nucleic acid in the sample (3) can be easily confirmed. Similarly, for example, the amount of the target nucleic acid in the sample (3) can be confirmed by comparing the amount of sediment captured when the sample (3) is used with a positive control and a negative control.

In addition, using a sample (4) that may contain the target nucleic acid, using as a control the aggregate distribution on the bottom surface of the container obtained using a sample containing a known target nucleic acid at a different concentration. It is also possible to confirm the presence or concentration or concentration of the target nucleic acid in the sample (4), as described above, by comparing the sediment trapping distribution with the control.
In addition, if the difference in the sediment capture distribution due to the presence or absence of the target nucleic acid and the difference in concentration is confirmed and recorded, a sample that may contain the target nucleic acid can be obtained without using the control. The presence or concentration of the target nucleic acid in the sample can be confirmed simply by subjecting it to detection. By doing in this way, a detection process can be simplified.

Further, when a plurality of types of target nucleic acids are contained in the sample, for example, by using different first and second ligands for each type of target nucleic acid, If only the microparticles to which the first and second receptors that specifically bind to the first and second ligands are bound are subjected to mixing, the one kind of target nucleic acid can be selectively detected. Similarly, depending on the type of target nucleic acid, microparticles bound with first and second receptors that specifically bind to the first and second ligands in the target nucleic acid are prepared for each target nucleic acid. If these fine particles are used for mixing, two or more kinds of target nucleic acids can be detected simultaneously.
Thus, the nucleic acid to be detected can be detected alone or a plurality of nucleic acids to be detected can be detected simultaneously.

  Here, as described above, the first and second ligands in the target nucleic acid may all be different for each type of target nucleic acid, as described above, but one of the first and second ligands, It may be common among a plurality of types of nucleic acids. By doing in this way, a detection process can be simplified.

  In the detection method described above, two types of ligands are used for one type of target nucleic acid, but three or more types of ligands may be used. In this case, fine particles to which a plurality of receptors that specifically bind to the ligand are bound may be prepared for each type of ligand.

(Container used for detection of target nucleic acid)
The container of the present invention used for detection of the target nucleic acid will be described in detail below. In the present invention, the container may be used alone, but a plurality of the containers provided on the same plate can be used. Specifically, for example, a 96-well microplate using the container as a well can be mentioned. However, of course, the number of wells is not limited to 96. When such a plurality of containers are provided, the capacity of one container is preferably 0.1 to 0.5 ml.

The bottom of the container of the present invention is provided with a site for capturing sediments, and the aggregates formed by linking the target nucleic acid and the fine particles and the sediments other than the aggregates more easily and with high accuracy. Can be identified. The sediment trapping part will be described in detail. For example, a plurality of concavities and convexities 42 having a sawtooth shape in the radial direction are concentrically arranged on a bottom surface 41 having a mortar shape as shown in FIG. Can be mentioned. Here, the length (h) of the first inclined surface connecting the peaks and valleys from the center 43 of the bottom surface to the outside of the unevenness 42 is preferably 0.2 to 20 μm, and from the center 43 to the outside. The length (l) of the second inclined surface connecting the valley and the mountain is preferably 0.5 to 50 μm, and the angle (θ ₁ ) formed by the straight line connecting the mountain and the center 43 with the horizontal plane is 15 to 45. It is preferable to be °. Moreover, it is preferable that the number of the unevenness | corrugations 42 is 10-50.

Moreover, as another specific example of the sediment capturing part, for example, as shown in FIG. 5, a bottom part 51 having a mortar shape of the container 50 is provided with a plurality of recessed parts 52 independently. Here, the angle (θ ₂ ) between the bottom surface 51 and the horizontal plane is preferably 15 to 45 °. More specifically, θ ₂ is an angle formed by a straight line connecting an arbitrary point excluding the recess 52 on the inclined surface of the bottom surface 51 and the center of the bottom surface 51 with the horizontal plane.
And this recessed part 52 is substantially hemispherical, it is preferable that the diameter (L) of the opening part is 0.05-100 micrometers, and it is preferable that the depth (d) is 0.03-60 micrometers.
The shape of the recess 52 is not limited to the substantially hemispherical shape shown here, as long as it can accommodate a plurality of the first fine particles and / or the second fine particles, preferably about 3-7. Different shapes and sizes may be used. The number of the recesses 52 is preferably about several tens to several hundreds.

The aforementioned size and shape of the sediment trapping part have been found in particular so that the aggregate and the sediment other than the aggregate can be easily and accurately distinguished. The target nucleic acid can be detected easily and with high accuracy by using a container equipped with a stable sediment trap. Therefore, when the range is out of the range, the sediment distribution pattern of the sediment may be difficult to distinguish between the aggregate and the sediment other than the aggregate.
Further, by using the containers shown in FIGS. 4 and 5 together, the target nucleic acid can be detected with higher accuracy.

The sediment capture distribution when the container 40 having the sediment capture section as shown in FIG. 4 is used will be described with reference to FIG.
When the target nucleic acid is not contained in the sample mixed with the first microparticle 33 and the second microparticle 34 (negative), as shown in FIGS. 6A and 6B, the target nucleic acid is not linked. The first fine particles 33 and the second fine particles 34 which have not been involved settle on the bottom surface 41, and most of them get over the irregularities 42 and reach the vicinity of the bottom bottom. On the other hand, when the target nucleic acid is contained (positive), as shown in FIGS. 6C and 6D, the first microparticle 33 and the second microparticle 34 are interposed via the target nucleic acid as described above. Connected together, agglomerates form and settle. Since the agglomerates are large in size, the aggregates remain as they are on the bottom surface 41 without getting over the irregularities 42 and are thinly deposited over the entire bottom surface 41. When the amount of target nucleic acid is large, the amount of aggregates deposited increases, and when the amount is small, the amount of sediments decreases. Therefore, the color density of the bottom surface of the container changes according to the target nucleic acid concentration in the sample. 6, (a) and (c) are longitudinal sectional views of the container 40 in a state where aggregates and / or sediments other than the aggregates are captured in the sediment capturing part, (b) ) And (d) are views when the bottom surface of the container 40 is viewed from above.

Next, the sediment capture distribution in the case where the container 50 having the bottom surface as shown in FIG. 5 is used will be described with reference to FIG.
When the target nucleic acid is not contained in the sample mixed with the first microparticle 33 and the second microparticle 34 (negative), as shown in FIGS. 7A and 7B, the target nucleic acid is not linked. The first fine particles 33 and the second fine particles 34 that were not involved settle on the bottom surface 51, most of them are captured by the recess 52, do not reach the vicinity of the bottom of the bottom surface 51, and the recess 52 of the bottom surface 51 It is held over almost the entire surface of the provided area. On the other hand, when the target nucleic acid is contained (positive), as shown in FIGS. 7 (c) and (d), the first microparticle 33 and the second microparticle 34 are interposed via the target nucleic acid as described above. Connected together, agglomerates form and settle. Since the aggregate is large in size, most of the aggregate reaches the vicinity of the bottom of the bottom surface 51 without being captured by the recess 52. If the amount of the target nucleic acid is large, the amount of aggregates deposited near the bottom of the bottom surface 51 increases and the area covering the bottom surface 51 increases. If the amount of target nucleic acids is small, the amount of aggregates deposited near the bottom of the bottom surface 51 decreases. And the area covering the bottom surface 51 is reduced. Accordingly, the color density and region of the discoloration site on the bottom surface of the container change according to the target nucleic acid concentration in the sample. 7, (a) and (c) are longitudinal sectional views of the container 50 in a state in which aggregates and / or sediments other than the aggregates are captured by the sediment capturing part, (b) ) And (d) are views when the bottom surface of the container 50 is viewed from above.

  In this way, the sediment capture distribution on the bottom surface of the container changes depending on the presence or absence or concentration of the target nucleic acid in the sample to be measured, so that the target nucleic acid can be detected easily and with high accuracy.

  Further, for example, the container of the present invention as shown in FIG. 4 or 5 can be manufactured by a conventionally known technique such as mold molding.

[Second method]
(Receptor binding to magnetic fine particles and container bottom)
Next, a second method for detecting a target nucleic acid will be described.
First, the step of binding the second receptor that specifically binds to the second ligand in the target nucleic acid to the magnetic fine particles will be described.

Examples of the magnetic fine particles used include triiron tetroxide (Fe ₃ O ₄ ), diiron trioxide (γ-Fe ₂ O ₃ ), various ferrites, iron, manganese, nickel, cobalt, chromium and other metals or cobalt, Magnetic fine particles made of an alloy containing nickel, manganese, etc., or hydrophobic polymers such as polystyrene, polyacrylonitrile, polymethacrylonitrile, polymethyl methacrylate, polycapramide, polyethylene terephthalate, or polyacrylamide containing these magnetic fine particles inside , Polymethacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, poly (2-oxyethyl acrylate), poly (2-oxyethyl methacrylate), poly (2,3-dioxypropyl acrylate), poly (2,3-dioxypropyl) Meta relay ), Latex such as cross-linked hydrophilic polymer such as polyethylene glycol methacrylate, or copolymer of about 2-4 kinds of monomers of the polymer, gelatin, liposome, or the magnetic fine particles as latex, gelatin, liposome, etc. Examples thereof include particles immobilized on the surface.

The particle size of the magnetic fine particles is preferably 0.1 μm to 20 μm.
If the particle size is out of the dimension, the sediment distribution pattern of the sediment may be difficult to distinguish between the aggregate and sediment other than the aggregate.
Examples of the method of binding the second receptor for the second ligand in the target nucleic acid to the magnetic fine particles include a method of physically adsorbing the receptor or a method of chemically supporting the receptor.

Examples of the physical adsorption method include a method in which a receptor is directly adsorbed and immobilized on magnetic fine particles, and a receptor is chemically bound to other proteins such as albumin and then adsorbed and immobilized. A method is mentioned.
Examples of the chemical loading method include activation of various functional groups such as amino group, carboxyl group, mercapto group, hydroxyl group, aldehyde group, and epoxy group present on the surface of the magnetic fine particles, and various types on the receptor. A method of immobilizing a receptor directly on a magnetic fine particle by binding with a functional group, a method of immobilizing a magnetic fine particle and a receptor by chemically bonding via a spacer molecule, for example, to other proteins such as albumin Examples include a method in which the protein is chemically bonded to the magnetic fine particles after the receptor is chemically bonded. Here, as a method of chemically bonding, any conventionally known method may be applied.

Next, the step of binding the first receptor that specifically binds to the first ligand in the target nucleic acid to the bottom surface of the container will be described.
The container used here may be any of transparent, translucent, colored and the like. Examples of the material include polystyrene, polypropylene, Teflon (registered trademark), polyethylene, methylpentene resin (TPX), plastics such as fluororesin, acrylic resin, polycarbonate, polyurethane, and vinyl chloride resin. And what is necessary is just to shape | mold by conventionally well-known methods, such as metal mold | die shaping | molding.
As a method for binding the first receptor that specifically binds to the first ligand in the target nucleic acid to the bottom surface of the container, a method of binding the receptor to fine particles or magnetic fine particles can be applied. What is necessary is just to select suitably according to the kind of body.

  Examples of the receptor bound to the magnetic fine particles and the container include, but are not limited to, antibodies, lectins, streptavidin, nickel, and the like. Nickel can be used because it has chelating ability.

  In this method, since at least two types of ligands are bound to the target nucleic acid, at least two types of receptors are prepared for one type of target nucleic acid. Then, a container 80 (FIG. 8) in which a first receptor 331 that specifically binds to the first ligand 31 is bound to the bottom surface, and a second receptor 341 that specifically binds to the second ligand 32. 9 is prepared.

  In the present invention, two or more kinds of receptors may be bound to one magnetic fine particle and a container. In this embodiment, among two or more types of ligands in one target nucleic acid, a receptor that specifically binds to one type of ligand and a receptor other than a receptor that specifically binds to the remaining types of ligands. , Bonded to one magnetic fine particle or container. That is, it is designed so that only one of two or more kinds of ligands in the target nucleic acid binds to one magnetic fine particle or container.

(Adding a sample that may contain the target nucleic acid and the magnetic fine particles to the container)
Next, a step of adding a sample that may contain a target nucleic acid and the magnetic fine particles will be described.
In this step, the sample that may contain the target nucleic acid and the magnetic fine particles may be mixed and then the mixture may be added to the container. The sample that may contain the target nucleic acid and the magnetic fine particles may be added. May be added to the container at the same time, but preferably, after adding a sample possibly containing the target nucleic acid to the container, the magnetic fine particles are added to the container. The sample and magnetic fine particles that may contain the target nucleic acid are preferably used as solutions. In this case, it is preferable to use a buffer solution, and a preferable buffer solution is, for example, PBS, but may be appropriately selected according to the situation.

  When the amount of the magnetic fine particles to be added to the container is too large, the detection accuracy decreases. Therefore, it is preferable that the amount of the magnetic fine particles is smaller than the target nucleic acid. Specifically, for the target nucleic acid of tens to hundreds of nM A fine particle amount of 0.01 to 0.5 w / v% is more preferable. And the number of the 1st receptors couple | bonded with the container bottom face should just be larger than the number of the 1st ligands in a target nucleic acid, and it is preferable that it is about 1.2 to 2 times the number of ligands.

  If the sample that may contain the target nucleic acid to be used for detection actually contains the target nucleic acid 30 as shown in FIG. 10 (a), as shown in FIG. 10 (b), the first The target nucleic acid 30 is linked to the bottom surface 81 of the container 80 through the binding between the ligand 31 and the first receptor 331, and as shown in FIG. 10 (c), the second ligand 32 and the second receptor The target nucleic acid 30 and the magnetic microparticle 90 are linked through the binding to 341. That is, the container 80 and the magnetic fine particle 90 are connected via the target nucleic acid 30. If the sample to be detected does not contain the target nucleic acid, such binding does not occur, and the magnetic microparticle 90 is not connected to the container 80.

(Process to observe the distribution of magnetic fine particles on the bottom of the container)
Subsequently, by applying a magnetic material such as a magnet to the bottom of the container from the outside of the container, the distribution pattern of the magnetic fine particles when the magnetic force is applied to the magnetic fine particles in the container is observed using a technique such as the naked eye or image processing. To do. For image processing and the like, the same method as described above may be applied.

As a result, when the target nucleic acid is contained in the sample (positive), as shown in FIGS. 11A to 11C, the container 80 Since the magnetic fine particles 90 are linked via the target nucleic acid 30, the magnetic fine particles 90 cannot be collected, and the magnetic fine particles 90 are held over almost the entire bottom surface 81 of the container 80. If the amount of the target nucleic acid 30 is large, the magnetic fine particles 90 that cover the bottom surface 81 of the container 80 increase, and if the amount is small, the magnetic fine particles 90 that cover the bottom surface 81 of the container decrease, so depending on the concentration of the target nucleic acid 30 in the sample, The color intensity of the bottom surface 81 changes. On the other hand, when the target nucleic acid is not contained in the sample (negative), the magnetic particles 90 are collected in the vicinity of the magnetic material on the bottom surface 81 by the magnetic material, as shown in FIGS.
As described above, the distribution pattern of the magnetic fine particles on the bottom surface of the container changes depending on the presence or absence or concentration of the target nucleic acid in the sample to be measured, so that the target nucleic acid can be detected easily and with high accuracy. 11, (a) and (b) are longitudinal sectional views of the container 80 in a state where the magnetic fine particles 90 are connected to the container 80, and (d) and (e) in FIG. The longitudinal cross-sectional view of the container 80 in the state which is not connected with 80, (c) and (f) is a figure when the bottom face of the container 80 is seen from upper direction.

  For example, when an antigen and an antibody are used as the ligand-receptor combination, this step is preferably performed at 0 to 37 ° C. for 5 to 30 minutes. In the case of other ligand-receptor combinations, an appropriate temperature, time, and buffer solution may be appropriately selected according to the type of the combination.

Next, a specific method for detecting a target nucleic acid will be described in detail.
Using a sample (1) containing a target nucleic acid with a known concentration to which a ligand is bound as described above, the target nucleic acid is added to the container according to the method described above, and magnetic fine particles are added to the container. The magnetic fine particle distribution pattern when a magnetic force is applied is confirmed and used as a positive control. Further, the sample (2) not containing the target nucleic acid is added to the container according to the method described above, the magnetic fine particles are added to the container, and the distribution pattern of the magnetic fine particles when a magnetic force is applied is confirmed. Use as negative control. Then, the sample (3) that may contain the target nucleic acid is similarly added to the container according to the above-described method, the magnetic fine particles are added to the container, and the distribution pattern of the magnetic fine particles on the bottom of the container is determined. The presence or concentration of the target nucleic acid in the sample (3) is confirmed by comparing with the positive control and the negative control.

Also, using a sample (4) that may contain the target nucleic acid, using the distribution pattern of the magnetic fine particles on the bottom surface of the container obtained by using a sample containing a known different concentration of the target nucleic acid as a control. It is also possible to confirm the presence or absence or concentration of the target nucleic acid in the sample (4) by comparing the distribution pattern of the magnetic fine particles with the control.
In addition, if the difference in the distribution pattern of the magnetic fine particles due to the presence or absence of the target nucleic acid or the difference in concentration is recorded and recorded, a sample that may contain the target nucleic acid can be obtained without using the control. The presence or concentration of the target nucleic acid in the sample can be confirmed simply by subjecting it to detection. By doing in this way, a detection process can be simplified.

Further, when a plurality of types of target nucleic acids are contained in the sample, for example, by using different first and second ligands for each type of target nucleic acid, Magnetic fine particles in which a first receptor that specifically binds to the first ligand is bound to a second receptor that specifically binds to the second ligand in the target nucleic acid. If only is added, the one kind of target nucleic acid can be selectively detected. Similarly, the first receptor that specifically binds to the first ligand in the plurality of types of target nucleic acids binds specifically to the second ligand in the plurality of types of target nucleic acids. If all the magnetic fine particles to which the second receptor is bound are added, the plurality of types of target nucleic acids can be detected simultaneously.
Thus, the nucleic acid to be detected can be detected alone or a plurality of nucleic acids to be detected can be detected simultaneously.

  Here, as described above, the first and second ligands in the target nucleic acid may all be different for each type of target nucleic acid, as described above, but one of the first and second ligands, It may be common among a plurality of types of nucleic acids. By doing in this way, a detection process can be simplified.

  In the detection method described above, two types of ligands are used for one type of target nucleic acid, but three or more types of ligands may be used. In this case, a receptor that specifically binds to each ligand is used. Among these three or more receptors, those that bind to the container and those that bind to the magnetic fine particles can be appropriately selected according to the purpose. good.

(Container used for detection of target nucleic acid)
The container used in this step is, for example, the one shown in FIG. 8 in consideration of the ease with which the magnetic fine particles not connected to the container are gathered when a magnetic force is applied to the magnetic fine particles. That is, it is preferable that the bottom surface 81 is substantially hemispherical and the angle (θ ₃ ) between the bottom surface 81 and the horizontal plane is 15 to 45 °. The bottom surface 81 may have a substantially conical shape other than the substantially hemispherical shape shown here. More specifically, θ ₃ is an angle formed by a straight line connecting an arbitrary point on the inclined surface of the bottom surface 81 and the center of the bottom surface 81 with the horizontal plane.
And like a 1st method, although such a container may be used independently, what provided the said container on the same plate can also be used. Specifically, for example, a 96-well microplate using the container as a well can be mentioned. However, of course, the number of wells is not limited to 96. When such a plurality of containers are provided, the capacity of one container is preferably 0.1 to 0.5 ml. The container used in the second method may be formed by bonding the receptor to the bottom of the container molded by a conventionally known method such as mold molding.

  Except for the points described above with respect to the second method, the same method as the first method can be applied to the second method.

  In the present invention, the target nucleic acid can be detected with higher accuracy by using the first technique and the second technique in combination. In the first method, as described above, the target nucleic acid can be detected with even higher accuracy by using the containers shown in FIGS. 4 and 5 together.

  As described above in detail, according to the method for detecting a target nucleic acid of the present invention, the nucleic acid to be detected can be detected easily at low cost with high accuracy. Thereby, for example, it is effective for cost reduction and simplification of a clinical test.

  The target nucleic acid detection method of the present invention can be used, for example, as a means for detecting a single nucleotide polymorphism (SNP) of a nucleic acid to be detected. In this case, when applying the PCR method as a method for obtaining a target nucleic acid to which a plurality of different haptens are bound, for example, a PCR-SSP method (Polymerase Chain Reaction-Sequence Specific Primer method) can be suitably used. The PCR-SSP method is designed to match the SNP site with the 3 ′ end of the primer by designing the sequence so that the 3 ′ end of one of the two types of primers overlaps the SNP site in the nucleic acid to be detected. Is a method for controlling amplification by PCR.

Hereinafter, further details of the principle will be described.
As a primer to which the first ligand is bound, for example, a primer in which digoxigenin (DIG) is bound to the 5 ′ end of a primer consisting of the base sequence shown in SEQ ID NO: 2, or a primer consisting of the base sequence shown in SEQ ID NO: 3 A primer having FITC bound to the end and a primer having biotin bound to the 5 ′ end of the primer consisting of the base sequence shown in SEQ ID NO: 4 are prepared. Next, an amplification reaction is performed on the nucleic acid to be detected having the base sequence shown in SEQ ID NO: 5 by the PCR-SSP method. At this time, as described as r in the base sequence shown in SEQ ID NO: 5, when the SNP site of the sample has two types of base A (adenine) and base G (guanine) as allyl, the allyl type is A / A homo, A / G hetero, and G / G homo.
When the sample SNP site is A / A homozygous, the DIG-labeled primer of SEQ ID NO: 2 matches the SNP site, and the nucleic acid to be detected is amplified by PCR together with the biotin-labeled primer of SEQ ID NO: 4. Is called. Since amplification is not performed with the FITC-labeled primer of SEQ ID NO: 2, the amplification product becomes a double-stranded DNA (target nucleic acid 1) in which DIG and biotin are separately bound to both 5 ′ ends.
In the same way, when the SNP site of the sample is G / G homozygous, the PCR product becomes double-stranded DNA (target nucleic acid 2) in which FITC and biotin are separately bound to both 5 ′ ends.
Furthermore, when the sample SNP site is A / G heterozygous, an amplification product in which the target nucleic acid 1 and the target nucleic acid 2 are mixed is obtained. By detecting the presence or absence of amplification of these nucleic acids, SNP typing becomes possible.
Based on the above-described principle, when detecting a SNP of a nucleic acid, it becomes possible to obtain a target nucleic acid modified with a hapten corresponding to the polymorphism.
Furthermore, it becomes possible to detect a plurality of SNPs of the nucleic acid to be detected. In this case, a primer to which a different type of hapten is bound for each different SNP site is prepared, and the nucleic acid to be detected may be amplified by the PCR-SSP method according to the above-described method.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
Here, an example of SNP typing based on the above-described principle is shown.
[Experiment 1]
<Extraction of DNA (nucleic acid to be detected)>
DNA was extracted from human blood. Specifically, a nucleic acid automatic extractor “Magulation System 6CG (manufactured by Precision System Science Co., Ltd.)” was used, and the use procedure was in accordance with the instruction manual for the product.
(1) A 5 ml blood sample of the subject was collected.
(2) 100 μl of whole blood collected was dispensed into a screw cap type 1.5 ml tube.
(3) The apparatus was started up, the extraction reagent and the specimen were set, and the machine was operated according to the operation program to start DNA extraction. As the extract, TE (10 mM Tris-HCl (pH 8.0 or 7.5) +1 mM EDTA) was used.
(4) After the extraction, the absorbance was measured using a spectrophotometer “nano drop (manufactured by Nanodrop, USA)”, and the concentration after extraction was measured.
Blood was collected from 9 subjects whose target SNP allyl type was known in advance by the above-described method, and DNA extraction was performed. As a result, 100 μl of genomic DNA having a concentration shown in Table 1 was obtained from 100 μl of whole blood.

<Amplification of detection target nucleic acid to which ligand is bound>
A primer having DIG bound to the 5 ′ end of the primer consisting of the base sequence shown in SEQ ID NO: 2, a FITC bound to the 5 ′ end of the primer consisting of the base sequence shown in SEQ ID NO: 3, the base shown in SEQ ID NO: 4 A nucleic acid to be detected having a base sequence shown in SEQ ID NO: 5 containing SNP was amplified by PCR using the genomic DNA as a template, using each of the primers consisting of the sequence having biotin bound to the 5 ′ end. .
(1) The concentration of genomic DNA was adjusted with TE so as to be 20 ng / μl.
(2) As shown in Table 2, reagents were prepared for the specimen.
(3) 20 μl of the prepared reagent was dispensed into a PCR tube.
(4) 1 μl of the sample solution was added to the PCR tube of (3).
(5) The PCR tube was set in a thermal cycler “Gene AmpPCRsystem 9700 (manufactured by Applied Biosystems)”, and PCR reaction was performed under the conditions shown in Table 3.

By the above method, the amplification reaction of the target nucleic acid was performed on the subjects of subjects 1 to 9.
In addition, a nucleic acid consisting of the base sequence shown in SEQ ID NO: 6 is bound to DIG at the 5 ′ end, and a nucleic acid consisting of the base sequence shown in SEQ ID NO: 7 is bound to biotin at the 5 ′ end. A heavy chain artificially synthesized target nucleic acid (I) was prepared, and FITC was bound to the 5 ′ end of the nucleic acid consisting of the base sequence shown in SEQ ID NO: 8, and 5 ′ of the nucleic acid consisting of the base sequence shown in SEQ ID NO: 9. A double-stranded artificial synthetic target nucleic acid (II) consisting of biotin bonded to the terminal is prepared, and these artificial synthetic target nucleic acids (I) and (II) are mixed to produce an artificial synthetic target nucleic acid (I) And the concentration was adjusted with TE so that (II) was 100 nM, respectively, and used as a positive control. TE was used as a negative control.

<Detection of target nucleic acid>
The target nucleic acid to which the ligand was bound prepared in the above step was detected.
The procedure and detection result of each detection method are shown below.
[Example 1]
The target nucleic acid was detected using the container shown in FIG.
(1) A solution of latex particles A (particle diameter: 0.1 μm, made by Celadin) sensitized with an anti-DIG antibody (manufactured by DAKO, catalog No: D5105) is buffered (200 mM MOPSO (pH 7.4)), The container a (h; 0.2 μm, l; 0.5 μm, θ ₁ ; 30) shown in FIG. 4, which was diluted with 4% dextran, 100 mM NaCl, 0.1% sodium azide) and formed by molding. 50 μl per well was dispensed into a 96-well reaction plate with 0).
(2) Anti-biotin antibody (manufactured by DAKO, catalog No: M0743) -sensitized latex particle solution was diluted with the buffer solution, and dispensed into the 96-well plate by 50 μl per well.
(3) 20 μl each of the test sample, positive control and negative control were added and well dispersed.
(4) After standing at room temperature for 5 minutes, compared with positive control and negative control, the distribution of capture of the sediment of the specimen on the bottom of the container was observed to determine positive or negative.

[Example 2]
Instead of the solution of latex particle A sensitized with anti-DIG antibody, the solution of latex particle B (particle size 10 μm, manufactured by Magsphere) sensitized with anti-FITC antibody (manufactured by DAKO, catalog No: D5101) Except that a 96-well reaction plate having wells of a container b (h; 20 μm, l; 50 μm, θ ₁ ; 30 °) shown in FIG. The target nucleic acid was detected in the same manner as in Example 1.

[Example 3]
The target nucleic acid was detected using the container shown in FIG.
Other than using a 96-well reaction plate in which the container c (L; 0.05 μm, d; 0.03 μm, θ ₂ ; 30 °) shown in FIG. 5 was used instead of the container a. Detected the target nucleic acid in the same manner as in Example 1.

[Example 4]
Instead of the solution of latex particle A sensitized with anti-DIG antibody, a solution of latex particle B sensitized with anti-FITC antibody (manufactured by DAKO, catalog No: D5101) was used, and a mold was used instead of container c In the same manner as in Example 3 except that a 96-well reaction plate having wells as containers d (L; 100 μm, d; 60 μm, θ ₂ ; 30 °) shown in FIG. Nucleic acid was detected.

[Example 5]
The target nucleic acid was detected using the container shown in FIG.
(1) An anti-biotin antibody was bound to the bottom of a 96-well plate “Immobilizer Amino Rockwell Module Plate (Nunc, Catalog No. 436023)” according to a conventionally known method.
(2) 50 μl of the buffer solution was dispensed per well.
(3) The subject, the positive control and the negative control were dispensed in 20 μl each.
(4) The mixture was allowed to stand at room temperature for 5 minutes.
(5) A solution of magnetic fine particles “Dynabeads M-450 Epoxy (manufactured by DYNAL, Catalog No. DB14001)” bound with an anti-DIG antibody was dispensed 50 μl per well and well dispersed.
(6) The mixture was allowed to stand at room temperature for 5 minutes.
(7) A magnet was applied from the outside of the bottom surface of the plate, and compared with the positive control and negative control, the distribution pattern of the magnetic fine particles on the bottom surface of the container was observed by the naked eye or a technique such as image processing to determine positive or negative.

[Example 6]
Target nucleic acid was detected in the same manner as in Example 5 except that magnetic fine particles bound with anti-FITC antibody were used instead of magnetic fine particles bound with anti-DIG antibody.

(judgment result)
The detection results of Examples 1 to 6 and the comprehensive determination results based on the detection results are shown in Table 4. As a comprehensive determination, if at least three detection results among the determination results of Examples 1 to 6 are the same, the result is determined as a final typing result. And it was confirmed by this invention that a target nucleic acid can be detected with high precision.

  The present invention is suitable for simplifying clinical tests, reducing costs, and the like, and is useful in the medical field.

It is a conceptual diagram which shows the method of obtaining a target nucleic acid. It is a figure which shows the microparticles | fine-particles with which the receptor was couple | bonded. It is a conceptual diagram which shows microparticles | fine-particles when it couple | bonds with a target nucleic acid and produces aggregation. It is sectional drawing which shows an example of the container of this invention. It is sectional drawing which shows the other example of the container of this invention. It is a figure which shows an example of the sediment capture distribution at the time of using the container shown in FIG. It is a figure which shows an example of the sediment capture distribution at the time of using the container shown in FIG. It is sectional drawing which shows the other example of the container of this invention. It is a figure which shows the magnetic fine particle to which the receptor was couple | bonded. It is a conceptual diagram which shows the state which connected the container shown in FIG. 8, and the magnetic microparticles shown in FIG. 9 via the target nucleic acid. It is a figure which shows an example of the distribution pattern of the magnetic fine particle in FIG.

Explanation of symbols

30 ... target nucleic acid, 31 ... first ligand, 32 ... second ligand, 33 ... first microparticle, 331 ... first receptor, 34 ... second 341 ... second receptor

Claims (23)

A method for detecting a target nucleic acid, wherein a first ligand and a second ligand of different types are bound to a nucleic acid to be detected,
A first microparticle in which a plurality of first receptors that specifically bind to the first ligand are bound; and a second microparticle in which a plurality of second receptors that specifically bind to the second ligand are bound. A step of preparing a fine particle of
Mixing a sample that may contain the target nucleic acid, the first microparticles, and the second microparticles in a container having a sediment capturing part on the bottom surface;
After the mixing step, the target nucleic acid and the first microparticles and the first microparticles and the second microparticles linked via the binding between the first ligand and the first receptor and the binding between the second ligand and the second receptor, respectively. A method for detecting a target nucleic acid, comprising the step of observing a capture distribution of an aggregate comprising two fine particles and / or a precipitate of the first fine particles and the second fine particles on the bottom surface of the container. A method for detecting a target nucleic acid, wherein a first ligand and a second ligand of different types are bound to a nucleic acid to be detected,
Preparing a container in which a plurality of first receptors that specifically bind to the first ligand are bound to the bottom surface;
Preparing a magnetic fine particle in which a plurality of second receptors that specifically bind to the second ligand are bound;
Adding the sample that may contain the target nucleic acid and the magnetic fine particles in the container;
A method for detecting a target nucleic acid, comprising the step of observing the distribution of magnetic fine particles on the bottom surface of the container by applying a magnetic force to the magnetic fine particles from the outside of the container after the adding step.   A method for detecting a target nucleic acid, wherein the detection method according to claim 1 and the detection method according to claim 2 are both performed.   In the method for detecting a target nucleic acid according to any one of claims 1 to 3, an observation result when a sample (1) containing a target nucleic acid having a known concentration is used, and the target nucleic acid is contained. The observation result when using the non-sample (2) and the observation result when using the sample (3) that may contain the target nucleic acid are compared. A method for detecting a target nucleic acid, comprising confirming the presence or concentration of the target nucleic acid.   In the method for detecting a target nucleic acid according to any one of claims 1 to 3, an observation result when a sample containing a target nucleic acid having a different concentration is used, and the target nucleic acid is contained. A method for detecting a target nucleic acid, comprising comparing the observation result when using a possible sample (4) and confirming the presence or concentration of the target nucleic acid in the sample (4).   The ligand is a hydrophilic organic compound, digoxigen, fluorescein, alexa, fluorescein isothiocyanate (FITC), 2,4-dinitrophenol (DNP), tetramethylrhodamine (TAMRA), polyoxygen having 6 or more amino acid residues. It consists of a peptide, a sugar chain in which two or more monosaccharides are bound, biotin, protein, polyhistidine, HA, glutathione S-transferase (GST), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (Flag), and derivatives thereof. The method for detecting a target nucleic acid according to any one of claims 1 to 5, wherein the target nucleic acid is selected from the group.   The target nucleic acid is prepared by a polymerase chain reaction method using a first primer to which a first ligand is bound and a second primer to which a second ligand is bound. The method for detecting a target nucleic acid according to any one of claims 1 to 6.   While the target nucleic acid incorporates at least one nucleotide to which the first ligand is bound using the first primer, at least the nucleotide to which the second ligand is bound using the second primer. The method for detecting a target nucleic acid according to any one of claims 1 to 6, wherein the target nucleic acid is prepared by polymerase chain reaction while incorporating one.   The target nucleic acid is of a plurality of different types, and at least one of the first and second ligands in the target nucleic acid is different for each type of target nucleic acid. A method for detecting a target nucleic acid according to 1.   The method for detecting a target nucleic acid according to any one of claims 1 to 9, wherein the target nucleic acid is derived from a living body.   The target nucleic acid according to any one of claims 1 and 3 to 10, wherein the first and second microparticles have a particle size of 0.1 to 10 µm and are optically distinguishable from each other. Detection method.   The detection of the target nucleic acid according to any one of claims 1 to 11, wherein a plurality of the containers are provided on the same plate, and the capacity of each container is 0.1 to 0.5 ml. Method. The sediment trapping portion of the container is a conical shape in which a plurality of irregularities having a serrated cross section in the radial direction are arranged concentrically on a bottom surface forming a mortar shape, and connects a mountain to a valley from the center to the outside. The length (h) of the first inclined surface is 0.2 to 20 μm, the length (l) of the second inclined surface connecting the valley to the mountain from the center to the outside is 0.5 to 50 μm, 13. The method for detecting a target nucleic acid according to claim 1, wherein an angle (θ ₁ ) formed by a straight line connecting the center with the horizontal plane is 15 to 45 °. The sediment trapping part of the container is provided with a plurality of recesses independently on the bottom of the mortar shape, and the angle (θ ₂ ) between the bottom and the horizontal plane is 15 to 45 °. The method for detecting a target nucleic acid according to any one of claims 1 and 3-12.   The method for detecting a target nucleic acid according to claim 14, wherein the opening has a diameter (L) of 0.05 to 100 µm and a depth (d) of 0.03 to 60 µm.   A method for detecting a target nucleic acid, comprising performing both the detection method according to claim 13 and the detection method according to claim 14 or 15.   The method for detecting a target nucleic acid according to any one of claims 2 to 10 and 12, wherein the magnetic fine particles have a particle size of 0.1 to 20 µm. The bottom surface of the container has a substantially hemispherical shape or a substantially conical surface shape, and an angle (θ ₃ ) between the bottom surface and a horizontal plane is 15 to 45 °, wherein The method for detecting a target nucleic acid according to any one of the above. A container used in the method for detecting a target nucleic acid according to any one of claims 1 and 3-12,
A plurality of concavities and convexities having a serrated cross section in the radial direction are arranged concentrically on the bottom surface forming a mortar shape, and the length (h) of the first inclined surface connecting the mountain to the valley from the center to the outside is 0. .2 to 20 μm, the length (l) of the second inclined surface connecting the valley to the mountain from the center to the outside is 0.5 to 50 μm, and the angle between the straight line connecting the mountain and the center with the horizontal plane ( A container for detecting a target nucleic acid, wherein θ ₁ ) is 15 to 45 °. A container used in the method for detecting a target nucleic acid according to any one of claims 1 and 3-12,
A container for detecting a target nucleic acid, wherein a plurality of recesses are independently provided on a bottom having a mortar shape, and an angle (θ ₂ ) between the bottom and a horizontal plane is 15 to 45 °.   21. The target nucleic acid detection container according to claim 20, wherein the opening has a diameter (L) of 0.05 to 100 μm and a depth (d) of 0.03 to 60 μm. A container used in the method for detecting a target nucleic acid according to any one of claims 2 to 10, 12 and 17,
A target nucleic acid detection container, wherein the bottom surface has a substantially hemispherical shape or a substantially conical surface shape, and an angle (θ ₃ ) between the bottom surface and a horizontal plane is 15 to 45 °. A container for detecting a target nucleic acid, wherein a plurality of containers according to any one of claims 19 to 22 are provided on the same plate.

Images